Minocycline Administration Does Not Have an Effect on Retinal Ganglion Cell Survival in a Murine Model of Ocular Hypertension.

This study aims to investigate two key aspects in a mouse model of ocular hypertension (OHT): first, the time course of retinal ganglion cell (RGC) death and the parallel activation of caspase-3 (a-Casp3+ cells) to narrow the therapeutic window; and second, the effect of caspase-3 and microglia inhibition by minocycline on RGC rescue in this model. RGC loss after OHT induction was significant at day 7 and progressed to 30 days. However, anatomical RGC death was preceded by significant Casp3 activation on day 3. Microglial inhibition by minocycline did not alter the course of OHT or rescue RGCs but resulted in a decrease in a-Casp3+ cells and phagocytic and total microglia. Therefore, RGC death commitment occurs earlier than their loss of Brn3a expression, microglial cells do not exacerbate RGC loss, and while this death is primarily apoptotic, apoptosis inhibition does not rescue RGCs, suggesting that alternative death pathways play a role in glaucomatous injury.

Retinal response to systemic inflammation differs between sexes and neurons.

Background: Neurological dysfunction and glial activation are common in severe infections such as sepsis. There is a sexual dimorphism in the response to systemic inflammation in both patients and animal models, but there are few comparative studies. Here, we investigate the effect of systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) on the retina of male and female mice and determine whether antagonism of the NLRP3 inflammasome and the extrinsic pathway of apoptosis have protective effects on the retina. Methods: A single intraperitoneal injection of LPS (5 mg/kg) was administered to two months old C57BL/6J male and female mice. Retinas were examined longitudinally in vivo using electroretinography and spectral domain optical coherence tomography. Retinal ganglion cell (RGC) survival and microglial activation were analysed in flat-mounts. Retinal extracts were used for flow cytometric analysis of CD45 and CD11b positive cells. Matched plasma and retinal levels of proinflammatory cytokines were measured by ELISA. Retinal function and RGC survival were assessed in animals treated with P2X7R and TNFR1 antagonists alone or in combination. Results: In LPS-treated animals of both sexes, there was transient retinal dysfunction, loss of vision-forming but not non-vision forming RGCs, retinal swelling, microglial activation, cell infiltration, and increases in TNF and IL-1ß. Compared to females, males showed higher vision-forming RGC death, slower functional recovery, and overexpression of lymphotoxin alpha in their retinas. P2X7R and TNFR1 antagonism, alone or in combination, rescued vision-forming RGCs. P2X7R antagonism also rescued retinal function. Response to treatment was better in females than in males. Conclusions: Systemic LPS has neuronal and sex-specific adverse effects in the mouse retina, which are counteracted by targeting the NLRP3 inflammasome and the extrinsic pathway of apoptosis. Our results highlight the need to analyse males and females in preclinical studies of inflammatory diseases affecting the central nervous system.

Expert Revision of Key Elements for Clinical-Grade Production and Qualification of Perinatal Derivatives.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Signalling pathways and cell death mechanisms in glaucoma: Insights into the molecular pathophysiology.

Glaucoma is a complex multifactorial eye disease manifesting in retinal ganglion cell (RGC) death and optic nerve degeneration, ultimately causing irreversible vision loss. Research in recent years has significantly enhanced our understanding of RGC degenerative mechanisms in glaucoma. It is evident that high intraocular pressure (IOP) is not the only contributing factor to glaucoma pathogenesis. The equilibrium of pro-survival and pro-death signalling pathways in the retina strongly influences the function and survival of RGCs and optic nerve axons in glaucoma. Molecular evidence from human retinal tissue analysis and a range of experimental models of glaucoma have significantly contributed to unravelling these mechanisms. Accumulating evidence reveals a wide range of molecular signalling pathways that can operate -either alone or via intricate networks - to induce neurodegeneration. The roles of several molecules, including neurotrophins, interplay of intracellular kinases and phosphates, caveolae and adapter proteins, serine proteases and their inhibitors, nuclear receptors, amyloid beta and tau, and how their dysfunction affects retinal neurons are discussed in this review. We further underscore how anatomical alterations in various animal models exhibiting RGC degeneration and susceptibility to glaucoma-related neuronal damage have helped to characterise molecular mechanisms in glaucoma. In addition, we also present different regulated cell death pathways that play a critical role in RGC degeneration in glaucoma.

Neuroinflammation and gliosis in the injured and contralateral retinas after unilateral optic nerve crush.

The main purpose of this study is to analyze the effects of unilateral optic nerve crush in the gene expression of pro- and anti-inflammatory mediators, and gliosis markers in injured and contralateral retinas. Retinas from intact, unilaterally optic nerve injured or sham-operated C57BL/6J mice were analyzed 1, 3, 9 and 30 days after the surgery (n = 5/group and time point) and the relative expression of TGF-ß1, IL-1ß, TNF-α, Iba1, AQP4, GFAP, MHCII, and TSPO was analyzed in injured and contralateral using qPCR. The results indicated that compared with intact retinas, sham-operated animals showed an early (day 1) upregulation of IL-1ß, TNF-α and TSPO and a late (day 30) upregulation of TNF-α. In sham-contralateral retinas, TNF-α and TSPO mRNA expression were upregulated and day 30 while GFAP, Iba1, AQP4 and MHCII downregulated at day 9. Compared with sham-operated animals, in retinas affected by optic nerve crush GFAP and TSPO upregulated at day 1 and TNF-α, Iba1, AQP4 and MHCII at day 3. In the crushed-contralateral retinas, TGF-ß1, TNF-α, Iba1 and MHCII were upregulated at day 1. TSPO was upregulated up to day 30 whereas TGF-ß1 and Iba1 downregulated after day 9. In conclusion, both sham surgery and optic nerve crush changed the profile of inflammatory and gliosis markers in the injured and contralateral retinas, changes that were more pronounced for optic nerve crush when compared to sham.

Methods to Identify Rat and Mouse Retinal Ganglion Cells in Retinal Flat-Mounts.

The identification of distinct retinal ganglion cell (RGC) populations in flat-mounted retinas is key to investigating pathological or pharmacological effects in these cells. In this chapter, we review the main techniques for detecting the total population of RGCs and various of their subtypes in whole-mounted retinas of pigmented and albino rats and mice, four of the animal strains most studied by the scientific community in the retina field. These methods are based on the studies published by the Vidal-Sanz's laboratory.

Comparative Analysis of Retinal Organotypic Cultures and In Vivo Axotomized Retinas.

Retinal organotypic cultures (ROCs) are used as an in vivo surrogate to study retinal ganglion cell (RGC) loss and neuroprotection. In vivo, the gold standard to study RGC degeneration and neuroprotection is optic nerve lesion. We propose here to compare the course of RGC death and glial activation between both models. The left optic nerve of C57BL/6 male mice was crushed, and retinas analyzed from 1 to 9 days after the injury. ROCs were analyzed at the same time points. As a control, intact retinas were used. Retinas were studied anatomically to assess RGC survival, microglial, and macroglial activation. Macroglial and microglial cells showed different morphological activation between models and were activated earlier in ROCs. Furthermore, microglial cell density in the ganglion cell layer was always lower in ROCs than in vivo. RGC loss after axotomy and in vitro followed the same trend up to 5 days. Thereafter, there was an abrupt decrease in viable RGCs in ROCs. However, RGC somas were still immuno-identified by several molecular markers. ROCs are useful for proof-of-concept studies on neuroprotection, but long-term experiments should be carried out in vivo. Importantly, the differential glial activation observed between models and the concomitant death of photoreceptors that occurs in vitro may alter the efficacy of RGC neuroprotective therapies when tested in in vivo models of optic nerve injury.

Pan-retinal ganglion cell markers in mice, rats, and rhesus macaques.

Univocal identification of retinal ganglion cells (RGCs) is an essential prerequisite for studying their degeneration and neuroprotection. Before the advent of phenotypic markers, RGCs were normally identified using retrograde tracing of retinorecipient areas. This is an invasive technique, and its use is precluded in higher mammals such as monkeys. In the past decade, several RGC markers have been described. Here, we reviewed and analyzed the specificity of nine markers used to identify all or most RGCs, i.e., pan-RGC markers, in rats, mice, and macaques. The best markers in the three species in terms of specificity, proportion of RGCs labeled, and indicators of viability were BRN3A, expressed by vision-forming RGCs, and RBPMS, expressed by vision- and non-vision-forming RGCs. NEUN, often used to identify RGCs, was expressed by non-RGCs in the ganglion cell layer, and therefore was not RGC-specific. γ-SYN, TUJ1, and NF-L labeled the RGC axons, which impaired the detection of their somas in the central retina but would be good for studying RGC morphology. In rats, TUJ1 and NF-L were also expressed by non-RGCs. BM88, ERRß, and PGP9.5 are rarely used as markers, but they identified most RGCs in the rats and macaques and ERRß in mice. However, PGP9.5 was also expressed by non-RGCs in rats and macaques and BM88 and ERRß were not suitable markers of viability.

Insights and future directions for the application of perinatal derivatives in eye diseases: A critical review of preclinical and clinical studies.

Perinatal derivatives (PnD) are gaining interest as a source for cell-based therapies. Since the eye is easily accessible to local administration, eye diseases may be excellent candidates to evaluate novel therapeutic approaches. With this work, we performed a systematic review of published preclinical and clinical studies addressing PnD in the treatment of ocular diseases. We have set two specific objectives: (i) to investigate the current level of standardization in applied technical procedures in preclinical studies and (ii) to assess clinical efficacy in clinical trials. Hereto, we selected studies that applied amniotic membrane (hAM) and mesenchymal stromal cells derived from amniotic membrane (hAMSC), placenta (hPMSC), umbilical cord (hUC-MSC) and Wharton's Jelly (hUC-WJ-MSC), excluding those where cells were not transplanted individually, following a systematic PubMed search for preclinical studies and consultation of clinical studies on https://clinicaltrials.gov and https://www.clinicaltrialsregister.eu/. Our bibliographic search retrieved 26 pre-clinical studies and 27 clinical trials. There was a considerable overlap regarding targeted ocular structures. Another common feature is the marked tendency towards (i) locally administered treatments and (ii) the PnD type. In the cornea/ocular surface, hAM was preferred and usually applied directly covering the ocular surface. For neuroretinal disorders, intra-ocular injection of umbilical or placental-derived cells was preferred. In general, basic research reported favourable outcomes. However, due to lack of standardization between different studies, until now there is no clear consensus regarding the fate of administered PnD or their mode of action. This might be accountable for the low index of clinical translation. Regarding clinical trials, only a minority provided results and a considerable proportion is in "unknown status". Nevertheless, from the limited clinical evidence available, hAM proved beneficial in the symptomatic relief of bullous keratopathy, treating dry eye disease and preventing glaucoma drainage device tube exposure. Regarding neuroretinal diseases, application of Wharton's Jelly MSC seems to become a promising future approach. In conclusion, PnD-based therapies seem to be beneficial in the treatment of several ocular diseases. However, much is yet to be done both in the pre-clinical and in the clinical setting before they can be included in the daily ophthalmic practice.

Perinatal derivatives application: Identifying possibilities for clinical use.

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.

The retina of the lab rat: focus on retinal ganglion cells and photoreceptors.

Albino and pigmented rat strains are widely used in models to study retinal degeneration and to test new therapies. Here, we have summarized the main topographical and functional characteristics of the rat retina focussing on photoreceptors and retinal ganglion cells (RGCs), the beginning and end of the retinal circuitry, respectively. These neurons are very sensitive to injury and disease, and thus knowing their normal number, topography, and function is essential to accurately investigate on neuronal survival and protection.

Immune recognition of syngeneic, allogeneic and xenogeneic stromal cell transplants in healthy retinas.

BACKGROUND: Advanced therapies using adult mesenchymal stromal cells (MSCs) for neurodegenerative diseases are not effectively translated into the clinic. The cross talk between the transplanted cells and the host tissue is something that, despite its importance, is not being systematically investigated. METHODS: We have compared the response of the mouse healthy retina to the intravitreal transplantation of MSCs derived from the bone marrow in four modalities: syngeneic, allogeneic, xenogeneic and allogeneic with immunosuppression using functional analysis in vivo and histology, cytometry and protein measurement post-mortem. Data were considered significant (p < 0.05) after nonparametric suitable statistical tests. RESULTS: Transplanted cells remain in the vitreous and are cleared by microglial cells a process that is quicker in allotransplants regardless of immunosuppression. All transplants cause anatomical remodelling which is more severe after xenotransplants. Xeno- and allotransplants with or without immunosuppression cause macro- and microglial activation and retinal functional impairment, being xenotransplants the most detrimental and the only ones that recruit CD45+Iba1-cells. The profile of proinflammatory cytokines changes in all transplantation settings. However, none of these changes affect the retinal ganglion cell population. CONCLUSIONS: We show here a specific functional and anatomical retinal response depending on the MSC transplantation modality, an aspect that should be taken into consideration when conducting preclinical studies if we intend a more realistic translation into clinical practice.

Intraocular implants loaded with A3R agonist rescue retinal ganglion cells from ischemic damage.

Retinal ganglion cell (RGC) loss underlies several conditions which give rise to significant visual compromise, including glaucoma and ischaemic optic neuropathies. Neuroprotection of RGCs is a clinical well-defined unmet need in these diseases, and adenosine A3 receptor (A3R) activation emerges as a therapeutic pharmacological approach to protect RGCs. A porous biodegradable intraocular implant loaded with 2-Cl-IB-MECA (selective A3R agonist) was used as a strategy to protect RGCs. Drug-loaded PCL implants released 2-Cl-IB-MECA for an extended period and the released 2-Cl-IB-MECA limited glutamate-evoked calcium (Ca2+) rise in RGCs. Retinal thinning due to transient ischemia was not prevented by 2-Cl-IB-MECA-PCL implant. However, 2-Cl-IB-MECA-PCL implants decreased retinal cell death, promoted the survival of RGCs, preserved optic nerve structure and anterograde axonal transport. We further demonstrated that 2-Cl-IB-MECA-loaded PCL implants were able to enhance RGC function that was compromised by transient ischemia. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA released from the PCL implant, this can be envisaged a good therapeutic strategy to protect RGCs.

Neuroprotection and Axonal Regeneration Induced by Bone Marrow Mesenchymal Stromal Cells Depend on the Type of Transplant.

Mesenchymal stromal cell (MSC) therapy to treat neurodegenerative diseases has not been as successful as expected in some preclinical studies. Because preclinical research is so diverse, it is difficult to know whether the therapeutic outcome is due to the cell type, the type of transplant or the model of disease. Our aim here was to analyze the effect of the type of transplant on neuroprotection and axonal regeneration, so we tested MSCs from the same niche in the same model of neurodegeneration in the three transplantation settings: xenogeneic, syngeneic and allogeneic. For this, bone marrow mesenchymal stromal cells (BM-MSCs) isolated from healthy human volunteers or C57/BL6 mice were injected into the vitreous body of C57/BL6 mice (xenograft and syngraft) or BALB/c mice (allograft) right after optic nerve axotomy. As controls, vehicle matched groups were done. Retinal anatomy and function were analyzed in vivo by optical coherence tomography and electroretinogram, respectively. Survival of vision forming (Brn3a+) and non-vision forming (melanopsin+) retinal ganglion cells (RGCs) was assessed at 3, 5 and 90 days after the lesion. Regenerative axons were visualized by cholera toxin ß anterograde transport. Our data show that grafted BM-MSCs did not integrate in the retina but formed a mesh on top of the ganglion cell layer. The xenotransplant caused retinal edema, detachment and folding, and a significant decrease of functionality compared to the murine transplants. RGC survival and axonal regeneration were significantly higher in the syngrafted retinas than in the other two groups or vehicle controls. Melanopsin+RGCs, but not Brn3a+RGCs, were also neuroprotected by the xenograft. In conclusion, the type of transplant has an impact on the therapeutic effect of BM-MSCs affecting not only neuronal survival but also the host tissue response. Our data indicate that syngrafts may be more beneficial than allografts and, interestingly, that the type of neuron that is rescued also plays a significant role in the successfulness of the cell therapy.

Axonal Injuries Cast Long Shadows: Long Term Glial Activation in Injured and Contralateral Retinas after Unilateral Axotomy.

BACKGROUND: To analyze the course of microglial and macroglial activation in injured and contralateral retinas after unilateral optic nerve crush (ONC). METHODS: The left optic nerve of adult pigmented C57Bl/6 female mice was intraorbitally crushed and injured, and contralateral retinas were analyzed from 1 to 45 days post-lesion (dpl) in cross-sections and flat mounts. As controls, intact retinas were studied. Iba1+ microglial cells (MCs), activated phagocytic CD68+MCs and M2 CD206+MCs were quantified. Macroglial cell changes were analyzed by GFAP and vimentin signal intensity. RESULTS: After ONC, MC density increased significantly from 5 to 21 dpl in the inner layers of injured retinas, remaining within intact values in the contralateral ones. However, in both retinas there was a significant and long-lasting increase of CD68+MCs. Constitutive CD206+MCs were rare and mostly found in the ciliary body and around the optic-nerve head. While in the injured retinas their number increased in the retina and ciliary body, in the contralateral retinas decreased. Astrocytes and Müller cells transiently hypertrophied in the injured retinas and to a lesser extent in the contralateral ones. CONCLUSIONS: Unilateral ONC triggers a bilateral and persistent activation of MCs and an opposed response of M2 MCs between both retinas. Macroglial hypertrophy is transient.

Mechanisms implicated in the contralateral effect in the central nervous system after unilateral injury: focus on the visual system.

The retina, as part of the central nervous system is an ideal model to study the response of neurons to injury and disease and to test new treatments. During the last decade is becoming clear that unilateral lesions in bilateral areas of the central nervous system trigger an inflammatory response in the contralateral uninjured site. This effect has been better studied in the visual system where, as a rule, one retina is used as experimental and the other as control. Contralateral retinas in unilateral models of retinal injury show neuronal degeneration and glial activation. The mechanisms by which this adverse response in the central nervous system occurs are discussed in this review, focusing primarily on the visual system.

Ly6c as a New Marker of Mouse Blood Vessels: Qualitative and Quantitative Analyses on Intact and Ischemic Retinas.

Ly6c is an antigen commonly used to differentiate between classical and non-classical monocytes/macrophages. Here we show its potential as a marker of the mouse vasculature, particularly of the retinal vascular plexuses. Ly6c was immunodetected in several tissues of C57BL/6 mice using isolectin IB4 as the control of vasculature staining. In the retina, Ly6c expression was analyzed qualitatively and quantitatively in intact, ischemic, and contralateral retinas from 0 to 30 days after the insult. Ly6c expression was observed in all organs and tissues tested, with a brighter signal and more homogeneous staining than the IB4. In the retinas, Ly6c was well expressed, allowing a detailed study of their anatomy. The three retinal plexuses were morphologically different, and from the superficial to the deep one occupied 15 ± 2, 24 ± 7, and 38 ± 1.4 percent of the retinal surface, respectively. In the injured retinas, there was extravasation of the classically activated monocyte/macrophages (Ly6chigh) and the formation of new vessels in the superficial plexus, increasing the area occupied by it to 25 ± 1%. In the contralateral retinas, the superficial plexus area decreased gradually, reaching significance at 30 days, and Ly6c expression progressively disappeared in the intermediate and deep plexuses. Although the role of Ly6c in vascular endothelial cell function is still not completely understood, we demonstrate here that Ly6c can be used as a new specific marker of the mouse vasculature and to assess, qualitatively and quantitatively, vascular changes in health and disease.

Activation of adenosine A3 receptor protects retinal ganglion cells from degeneration induced by ocular hypertension.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness. This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatments available target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development. However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effective treatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A3 receptor (A3R) activation confers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation of A3R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterized animal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A3R agonist, abolished the alterations induced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a- and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A3R agonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved the structure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisage that A3R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage.